destroyed, or removed from the body. The experiments to be described intravenously injected insulin was observed in normal animals, in

Transcription

1 417 J. Physiol. (I940) 97, 4I I :6I2.I29.I ON THE DISAPPEARANCE FROM THE BLOOD OF INTRAVENOUSLY INJECTED INSULIN BY H. K. GOADBY1 AND J. S. RICHARDSON From the Medical Unit Laboratories, St Thomas's Hospital, London (Received 1 September 1939) IT is not yet universally agreed which of the two hypotheses about the secretion of insulin by the pancreas is correct; whether insulin is periodically secreted in response to a rise in the blood-sugar concentration; or whether there is a continuous secretion at a constant rate, a more recent view put forward by Soskin, Allweiss & Cohn [1934]. In either case, a certain amount of insulin is required daily by the body. Furthermore, when normal men or animals, or a diabetic patient, or an animal with experimentally produced diabetes, are injected with insulin, this insulin appears to work only for a limited time. It seems then that, whichever hypothesis as to the secretion of insulin is right, there is apparently a mechanism whereby insulin is either inactivated or destroyed, or removed from the body. The experiments to be described in this paper were designed to enquire into such a mechanism. Briefly, the apparent disappearance from the circulating blood of intravenously injected insulin was observed in normal animals, in animals with the liver excluded from the circulation, or with the kidneys so excluded, or after the injection of Young's glycotropic hormone of the pituitary. METHODS Rabbits of 1*5-2*8 kg. were used throughout. The experiments to observe the disappearance of insulin from the circulation of normal, intact rabbits and of operated animals, were performed in exactly the same way; the technique was as follows: I. The rabbit A, to be tested for insulin disappearance, was first given 10 g. glucose in 20 c.c. water by stomach tube in order to prevent hypoglycaemic reactions occurring as the result of the large dose of 1 Mackenzie Mackinnon Research Fellow, of the Royal College of Physicians, of London and the Royal College of Surgeons of England. PH. XcVII. 27

2 418 H. K. GOADBY AND J. S. RICHARDSON insulin to be given; 5-20 min. later it received an intravenous injection of 40 units of insulin (1 c.c. of Boot's "double strength" ordinary insulin). From time to time after this, successive 5 c.c. samples of blood were taken from an ear vein directly into three drops of 20% potassium oxalate solution in a small tube. The blood was mixed at once to prevent clotting and kept at room temperature. (Preliminary experiments showed that the hypoglycaemic action of the blood with insulin added in vitro, or of blood taken whilst insulin is circulating in vivo, is not diminished on keeping at room temperature or 37 for at least 6 hr., the maximum time it was kept before testing in any experiment.) II. Three other rabbits B, C and D, which had been starved hr., were placed in their boxes and allowed 5-20 min. in which to settle down. From each, three preliminary blood samples for sugar content were then taken at 10 min. intervals. After this, 5 c.c. of the first sample of oxalated blood from rabbit A were injected into the marginal ear vein of B; blood was then taken for blood-sugar estimation on B at 10 min. intervals for 50 min. The same process was repeated on rabbit C, with the second sample from A, and on D with the third sample. Thus the depression of the blood-sugars of rabbits B, C and D would indicate the presence of insulin in the blood samples from rabbit A, and therefore its presence in the circulation of A at stated times after it was injected intravenously. Blood sugars. These were estimated on single 0-1 c.c. samples of blood taken from a marginal ear vein by the method of Hagedorn & Jensen [1923]. Exclusion of the liver from the circulation. This was done by following exactly the technique of Himsworth [1938], 3 weeks being left between the first and second operations, 4 days between the second operation and the insulin experiment. Pituitary glycotropic hormone. This was kindly made by Dr F. G. Young, to whom the author is greatly indebted. Four c.c. of the sample provided were injected 18 hr., and 4 c.c. 2 hr. before the experiment, thus following exactly his recommended procedure [1936]. Exclusion of the kidneysfrom the circulation. A technique was evolved similar to the one used for exclusion of the liver. Under ether anaesthesia, with full aseptic precautions, the rabbit's abdomen is opened, the intestines are retracted to one side, and the kidney exposed to view. A ligature is ready, consisting of 2 in. of soft, round lamp-wick, to each end of which about 6 in. lengths of No. 18 thread are tied. By blunt dissection, the renal pedicle is exposed in the peritoneal fat, the ligature is slipped

3 REMOVAL OF BLOOD INSULIN 419 around the pedicle to include the artery and vein, but not the ureter, and is tied in a loose half hitch. With needles the threads are then passed, one through the posterior abdominal wall to come out through the skin about 2 in. from the midline as nearly as possible dorsal to the kidney, the other through the anterior abdominal wall, so that it lies directly ventral to the kidney after the incision is closed. The intestines having been retracted to the other side, a ligature is similarly passed round the other renal pedicle, and the two thread ends passed out through the abdominal wall. The abdomen is then closed in two layers, the peritoneum plus muscle and the skin, by continuous sutures. Each pair, right and left, of threads is tied together so as to lie easily round the side of the animal. The wound is covered with a dressing of gauze soaked in collodion. The threads are protected from chewing or rubbing by covering them with a piece of adhesive strapping right round the abdomen. In a warm cage the animals soon recover. Four days later, the insulin experiment is performed. The strapping is carefully removed, exposing the threads; each pair, dorsal and ventral, is untied or cut, and then pulled firmly and steadily until quite tight. The ends are then cut off. The animals seem quite undisturbed by this procedure. That the ligatures are tight around the renal pedicles and remain so, thus excluding the kidneys from the circulation, is proved by (i) the rabbits pass no urine until death; (ii) at post-mortem, the lamp-wick ligatures are tight on the renal pedicles and the kidney parenchyma is degenerated. Method of expressing results. The criterion had first to be established of what is the minimum drop in blood-sugar concentration in rabbits that can be taken as evidence that the injected blood has a hypoglycaemic action, and therefore presumably contains insulin. (For the sake of brevity in this paper this action of the blood samples in depressing the blood sugar will be called the insulin action.) The blood sugars of ten normal rabbits (18-20 hr. fasting) were estimated at 10 min. intervals for 80 min., the period of the standard experiment. These experiments, Fig. 1, showed that owing to considerable spontaneous variations in the blood-sugar concentrations obtained under these conditions and by this technique, insulin action of injected blood can only be inferred if the blood-sugar concentration falls in a smooth curve to more than 20 mg. below the lowest of the three pre-injection values. Therefore, a blood will be said to have given a " positive reaction ' only if this occurred; irregular drops or variations of less than 20 mg. are called negative. 27-2

4 420 H. K. GOADBY AND J. S. RICHARDSON The results are also expressed in the form of curves; in these, the third pre-injection blood sugars are taken as 0; all the other values in any one experiment are calculated as mg. plus or minus this reading. The points on the curves shown are the means of all experiments in a group; the shaded areas are drawn to include plus and minus the probable to : Fig. 1. Blood-sugar variations in normal, fasting rabbits. error of each man. This "probable error" is obtained from the formula 3 x lvnfa, where a is the standard deviation of the blood-sugar difference values, n the number of experiments; the factor 3 is taken in place of the more usual 2 because of the small size of the samples. RESULTS Preliminary. The first problem was to find out what is the minimum dose of actual insulin which gives a "positive reaction" in normal rabbits; the experiments are summarized in Table I. From these figures TABLE I Blood-sugar depressions Positive Insulin units reactions Negative reactions 1*0-0* (16 mg. fall) (13,, ) (19,, ) * Sensitivity of normal fasting rabbits to intravenous insulin. Blood-sugar reactions to diminishing doses. it may be concluded that the limit of sensitivity of the rabbits under the conditions and criteria of the experiments is about 0-03 unit. This means

5 REMOVAL OF BLOOD INSULIN 421 that if a negative reaction is obtained from a blood sample it contains probably less than 1 unit in the volume (5 c.c.) injected. A positive reaction indicates that more than I unit is present. From the blood-sugar depression curves obtained, it was found impossible by a single experiment on a rabbit to obtain anything approaching an accurate measure of the dose given, whatever the method of calculation used. This is, of course, to be expected in animal experiments, and is in agreement with the findings of all workers on the biological assay of insulin. Disappearance of insulin action from circulating blood In intact rabbits (twenty-one animals). Seventeen of these had blood taken immediately, i.e. 1-5 min. after insulin injection, eleven 30 min., eleven 60 min., and seven 90 min. later. Table II and curves 2, 3, 4 and 5, Fig. 2, show that in normals, blood taken immediately after the Immediately after insulin 30 min. after insulin 0 Blood ~~~~~~~~Blood g _,,,,injected injected ff0 MMil I a. [ 2 3J ~ min. after insulin 90 mi. after insuln 0-40~~~~~~~~~ tb 0 Blood injece ~~~~~~~~~Blood inijected 1hMin Fig. 2. Disappearance of insulin from blood in normal animal[s. Curves of blood-sugar depression following transfusion. Donors =.normal rabbits. Bloods taken 2, 30, 60 and 90 min. after 40 units insulin intravenously. injection of 40 units of insulin intravenously, has a strong insulin action. In the 30, 60 and 90 min. samples, this action gradually diminishes; at 90 min. all seven tests were negative.

6 422 H. K. GOADBY AND J. S. RICHARDSON TABLE II Times after insulin injection of taking blood samples A- Groups 2-5 min. 30 min. 60 min. 90 min. Normals 100 (17) 82 (11) 40 (10) 0 (7) Afterfirst operation on 100 (6) 80 (5) 17 (6) liver Livers excluded 100 (11) 73 (11) 57 (7) Kidneys excluded 100 (10) - 90 (10) 70 (10) Glycotropic hormone 100 (4) 50 (4) 25 (4) Insulin action of bloods taken at various times after injection of insulin in the various experimental groups. The main figures denote the percentage of "positive reactions" obtained, the figures in parentheses are the numbers of experiments in the groups. That this insulin action is due to the exogenous insulin, and not to endogenous insulin secreted in response to the glucose given, was controlled by experiments in which normal rabbits were simply given glucose by stomach tube, and their blood tested for insulin action afterwards. In one animal, blood was taken 2 min. after the glucose; this gave a just positive reaction; the figures of the blood-sugar depression of the rabbit on which this blood was tested were -7, - 14, -24, - 19, -10 mg. at successive 10 min. intervals after receiving the blood. By comparison with curve 2, Fig. 2, of the results immediately after injection of insulin, it will be seen that the points lie well outside the probable error of the mean depression curve. Two animals had blood taken 23 min. after glucose only; both gave negative results, as compared with nine positive out of eleven 30 min. after insulin. Two animals gave completely negative results 50 min. after glucose only. No further control experiments were done, as it was thought that the insulin concentration in the blood in response to glucose would be outside the limit of sensitivity of the method of testing for it. This supposition was strengthened by the facts that the one positive response to glucose only was so small, and that it was known that up to 1 hr. after giving the glucose, the blood sugars were at high enough levels to be adequate stimuli for the secretion of endogenous insulin; and yet after 23 and 50 min. all tests were negative. It can, therefore, be reasonably assumed that the insulin action of blood taken after intravenous injection of insulin, as tested by the present technique, is due to the exogenous insulin. After exclusion of the liver (11 rabits). The glucose was given first; after 5-10 min. the ligatures were pulled tight, and after 1-3 min. more, the insulin injected. When possible, blood was taken 2, 30 and 60 min. after the insulin. The tightening of the ligatures and the injection of insulin were followed by severe shock, so that in four cases only could

7 REMOVAL OF BLOOD INSULIN 423 the three blood samples be obtained from the ear vein. In three more, the third blood sample (60 min.) was taken direct from the heart after 60 min. after insulin Fig. 3. Blood-sugar depression. Donors=liver-excluded group. Blood injected m n4ii It I u 50 opening the chest, the rabbit being moribund. Four animals did not survive much longer than 30 min., and in two of these, heart blood was then taken. 0 This failure of the animals to survive very long after exclusion of the liver was disappointing in comparison with Hims- "'-20 worth's experiments, in which over 4 hr. _ was the average survival time. The rabbits 0 in the author's experiments appeared to o0 die of shock; only in one case was any ' other obvious cause found post-mortem, -20 namely haemorrhage from a torn hepatic E: vein. Five out of eleven animals in which the liver was excluded died in convulsions: W these were not due to hypoglyeaemia, 0. their blood sugars during convulsions were pa 119, 162, 138, 104, 91 mg./100 c.c. respec- 20 _ :,. tively. In five rabbits which died of shock injected and did not have convulsions, the blood -40 Mn sugars were 161, 49, 167, 88, 32. TableIIan cures 6,7 Fg. Table II and curves 3Fig. 4. Mean curves 6, 7 and from five rabbits 8, Fig. 3, before and after exclusion of the show that the disappearance of insulin liver. Top curve = results of bloods taken immediately after insulin. action proceeds at the same rate when the Middle curve =30 min. after inliver is excluded from the circulation as it insulin. sulin. Bottom Continuous curve=60min. line = before after does in the normal, intact animals: the liver exclusion. Dotted line = after differences in the figures are too small to liver exclusion. be statistically significant. On five rabbits the insulin experiment was

8 424 H. K. GOADBY AND J. S. RICHARDSON done both between the first and second operations, i.e. with the portal vein partly obstructed, and also when the liver was completely excluded from the blood stream. The results are given in Fig. 4. It may be thought that these curves indicate that the disappearance of insulin action is somewhat slower after exclusion of the liver: but the condition of shock of the animals, especially in view of the results after exclusion of the kidneys to be described later, makes it impossible to lay any stress on the somewhat small differences obtained. To sum up, there is no evidence from these experiments that exclusion of the liver from the circulation has any effect on the rate of disappearance of the hypoglycaemic action of the circulating blood after intravenous injection of insulin. +20 Immediately after insulin 30 mnu. after insulin 60 nii. after insulin 0 \ -20; ~-40- Blood Blood Blood injected injected injected -60oMin. Mm. I 1Min.1 J Fig. 5. Blood-sugar depression. Donors =glycotropic-hormone injected group (mean values only). Glycotropic hormone. The doses of the hormone given were assumed to be such as to render the rabbits insensitive to the hypoglycaemic action of at least 3 units of insulin intravenously. Evidence that the hormone was active in this respect was afforded by the fact that in all four rabbits the blood sugar rose markedly 30 min. after the glucose and insulin were given; this happened only in four out of ten normals, three out of nine with livers excluded, in none out of six with kidneys excluded: the action of insulin appeared thus to be inhibited to some extent. Only sufficient hormone for four animals was available; the results do not differ from those of normals, as seen in curves 9, 10 and 11, Fig. 5. In these only the means of the four experiments are plotted. After the exclusion of the kidneys (11 experiments). This group shows that the insulin action persists longer in the blood stream of animals in which the kidneys are excluded from the circulation, than in that of normal, intact animals. Of the animals so treated 70 % showed a

9 REMOVAL OF BLOOD INSULIN 425 positive insulin action 90 min. after injection of insulin. This is a statistically significant difference, as is also the number of positive reactions after 60 min. Table II and curves 12, 13 and 14, Fig. 6, illustrate this. The rabbits survived 1-4 days after the experiment; at the time of the insulin experiment they appeared quite normal and undisturbed. Immediately after insulin 60 min. after insulin 90 min. after insulin t4-40 -',, + Ḃlood + s Blood r Blood injected injected injected -60 IM@S 1 1 _l.n. 1- L_, in In I I I I Fig. 6. Blood.sugar depression. Donors=kidney-excluded group. Blood-sugar concentrations of the rabbits after glucose and insulin. The mean blood sugars of the various groups of insulin-injected rabbits are given in Table III. From these figures it can be seen that the glucose effectively prevents any hypoglycaemia during the period of the experiments, even in the animals with the livers tied off: in these the glucose can only be absorbed into the general blood stream via the collateral circulation established by the first operation. TABLE III Times after insulin injection 2 min. 30 min. 60 min. Normals 204 (39) 190 (78) 185 (76) Livers excluded 131 (30) 134 (61) 122 (68) Kidneys excluded 208 (26) 134 (39) 155 (41) Glycotropic hormone injected 231 (39) 286 (57) 280 (56) Blood sugars of rabbits after glucose by stomach tube and insulin intravenously; concentration in mg./100 c.c.; figures in parentheses= standard deviations. The blood sugars of the liver-excluded group lie at a consistently lower level than in the normals, whereas those of animals receiving glycotropic hormone are at a higher level. This may be a true finding and is a priori to be expected. However, on the criterion that for a difference between the means to be significant it must be greater than three times the standard error, the differences obtained can all be due to errors of sampling. The only exceptions are: (1) The liver-excluded rabbits

10 426 H. K. GOADBY AND J. S. RICHARDSON immediately after injection of insulin had lower blood sugars than all the other groups at the same time; this presumably indicates slower absorption of glucose into the general circulation, as the blood is taken after glucose has been given, before the insulin has had time to act. (2) The liver-excluded and the glycotropic-hormone injected groups show a considerable difference throughout; owing to the very small number in the latter group, no emphasis will be laid on this difference. DIscusSION Blood taken from a rabbit immediately after intravenous injection of 40 units of insulin depresses the blood sugar of normal fasted rabbits. This is in agreement with the findings of Heymans & Heymans [1927]; they found that blood taken within 4 min. of intravenous injections of 2-15 units of insulin occasionally produced hypoglycaemic reactions in other rabbits. Kepinow & Ladept-Petit Dutaillis [1927a], however, failed to demonstrate on dogs, any blood-sugar depressing action of 360 c.c. of blood from other dogs, taken at various times down to min. after the intravenous injection of 12 units of insulin; these doses of insulin are much smaller than those used by the authors. This insulin action, present immediately after injection of insulin, gradually diminishes, and by 90 min. has almost, if not quite vanished. Now a 2 kg. rabbit has about 200 c.c. of blood; also it has been shown above that the insulin action of the circulating blood in these experiments is due only to the injected insulin. Its concentration at the start can theoretically be 40 units in 200 c.c. (1 unit in 5 c.c.); in 90 min. this has fallen to less than 4 units in 200 c.c. ( 10 unit in 5 c.c.). The disappearance of the action is unaffected by exclusion of the liver from the circulation, a finding in contrast with that of Kepinow & Ladept-Petit Dutaillis [1927b]. These workers anastomosed the portal vein to the renal vein of a dog; with the liver thus removed from the circulation, they demonstrated insulin in the circulating blood, 3-20 min. after 12 units of insulin given intravenously. By comparison with their first experiment they concluded that the liver inactivates the insulin or removes it. However, the authors' experiments indicate that the liver is not necessary for the gradual disappearance of the insulin action of the circulating blood after a large dose of insulin is injected intravenously; glycotropic hormone seems also to have no effect on this disappearance. Exclusion of the kidneys, however, from the circulation results in a longer persistence of the insulin action in the blood; this suggests that the kidneys are responsible for the removal of insulin from the blood when it

11 REMOVAL OF BLOOD INSULIN 427 is present there in high concentration. Fisher & Noble [1923] in the early days of insulin asserted that they could recover from the urine 35 out of 40 units of subcutaneously injected insulin. Partos [1932], by an extraction method, obtained insulin from the urine of all subjects tested except human diabetics, pancreatectomized cats, and rabbits fasted for more than 48 hr. Burger & Friedman [1938] injected rabbits' urines intravenously into other rabbits. They found that there was no insulin action in normal rabbit urine; it was present there up to 2 hr. after the intravenous injection of 200 units of insulin. Recordier & Andriac [1935] found that bilateral nephrectomy resulted in the persistence up to 100 min. of the action of 2 units of insulin injected intravenously, whereas in the same rabbits before nephrectomy, its action was over by then Ṫhe amount of insulin circulating in the blood of a rabbit after 40 units of insulin are injected intravenously is probably far in excess of normal physiological limits; therefore the mechanism for inactivating or getting rid of this may not be the one which deals with insulin normally secreted by the pancreas. It is hoped with the aid of a more sensitive test animal to extend these observations to concentrations of insulin more nearly physiological, and safely attainable by injection of insulin in human beings. SUMMARY 1. After the injection of 40 units of insulin intravenously into rabbits, it is possible to show that the circulating blood has a bloodsugar depressing action, by transfusing it into other fasted rabbits; this action is shown to be due to the injected insulin. 2. This hypoglycaemic action gradually disappears and has gone by the end of about 90 min. 3. The rate of disappearance is not affected by excluding the liver from the circulation, nor by the previous injection of glycotropic hormone of the pituitary gland. 4. Excluding the kidneys from the circulation results in a persistence of the insulin action. 5. The liver, therefore, is not responsible for the inactivation or removal of large doses of exogenous insulin from the blood stream; the kidneys are concerned in such a mechanism.

12 428 H. K. GOADBY AND J. S. RICHARDSON REFERENCES Burger, M. & Friedman, B. [1938]. Proc. Soc. exp. Biol., N.Y., 38, 840. Fisher, F. & Noble, B. E. [1923]. Amer. J. Phy8iol. 67, 72. Hagedorn, H. C. & Jensen, B. N. [1923]. Biochem. Z. 135, 46. Heymans, F. & Heymans, C. [1927]. C.R. Soc. Biol., Pari8, 96, 719. Himsworth, H. P. [1938]. J. Phy8iol. 91, 413. Kepinow, L. & Ladept-Petit Dutaillis [1927a]. C.R. Soc. Biol., Pari8, 96, 25. Kepinow, L. & Ladept-Petit Dutaillis [1927b]. C.R. Soc. Biol., Paris, 96, 371. Partos, A. [1932]. Z. ges. exp. Med. 84, 374. Recordier, M. & Andriac, M. [1935]. Marseilles Med. 72, 741. Soskin, S., Allweiss, M. D. & Cohn, D. J. [1934]. Amer. J. Physiol. 109, 155. Young, F. G. [1936]. Lancet, ii, 237.